A single pair of leucokinin neurons are modulated by feeding state and regulate sleep–metabolism interactions

Dysregulation of sleep and feeding has widespread health consequences. Despite extensive epidemiological evidence for interactions between sleep and metabolic function, little is known about the neural or molecular basis underlying the integration of these processes. D. melanogaster potently suppress sleep in response to starvation, and powerful genetic tools allow for mechanistic investigation of sleep–metabolism interactions. We have previously identified neurons expressing the neuropeptide leucokinin (Lk) as being required for starvation-mediated changes in sleep. Here, we demonstrate an essential role for Lk neuropeptide in metabolic regulation of sleep. The activity of Lk neurons is modulated by feeding, with reduced activity in response to glucose and increased activity under starvation conditions. Both genetic silencing and laser-mediated microablation localize Lk-dependent sleep regulation to a single pair of Lk neurons within the Lateral Horn (LHLK neurons). A targeted screen identified a role for 5′ adenosine monophosphate-activated protein kinase (AMPK) in starvation-modulated changes in sleep. Knockdown of AMPK in Lk neurons suppresses sleep and increases LHLK neuron activity in fed flies, phenocopying the starvation state. Further, we find a requirement for the Lk receptor in the insulin-producing cells (IPCs), suggesting LHLK–IPC connectivity is critical for sleep regulation under starved conditions. Taken together, these findings localize feeding-state–dependent regulation of sleep to a single pair of neurons within the fruit fly brain and provide a system for investigating the cellular basis of sleep–metabolism interactions.


Introduction
Dysregulation of sleep and feeding has widespread health consequences, and reciprocal interactions between these processes underlie a number of pathologies [1][2][3][4]. Sleep loss correlates with increased appetite and insulin insensitivity, while short-sleeping individuals are more likely to develop obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease [1,3,4]. Although the neural basis for sleep regulation has been studied in detail, little is known about how feeding state and changes in metabolic function modulate sleep [5,6]. Understanding how sleep and feeding states are integrated may provide novel insights into the comorbidity of disorders linked to sleep and metabolic regulation.
Animals balance nutritional state and energy expenditure in order to achieve metabolic homeostasis [6,7]. In both flies and mammals, diet potently affects sleep regulation, supporting the notion that sleep and metabolic state interact [5,6,8]. Starvation leads to sleep loss or disrupted sleep architecture, presumably to induce foraging behavior, while high-calorie diets have complex effects on sleep depending on macronutrient content [9][10][11][12]. Behavioral and physiological responses to changes in feeding state are modulated both by cell-autonomous nutrient centers in the brain that detect changes in circulating nutrients and through communication between brain and peripheral tissues [13], yet the neural basis for the integration of sleep and feeding state remain poorly understood.
The fruit fly, D. melanogaster, provides a powerful model for investigating sleep regulation. Flies display all the behavioral hallmarks of sleep, including extended periods of behavioral quiescence, rebound following deprivation, increased arousal threshold, and species-specific changes in posture [14,15]. Many genetic mechanisms regulating sleep are conserved from flies to mammals. In addition, high-throughput systems for sleep analysis in Drosophila have led to the identification of both novel and highly conserved sleep genes [16,17]. Further, stimulants including caffeine, amphetamine, and cocaine have been shown to suppress sleep in flies [15,18,19]. Thus, at the molecular, pharmacological, and behavioral levels, flies provide a model for studying genetic regulation of mammalian sleep.
A number of genes and neurons that are required for the integration of sleep and feeding states have been identified, including core-circadian clock genes, metabolic hormones, and sensory neurons [9,[20][21][22]. While many identified regulators of sleep-metabolism interactions broadly impact these processes [6], a mutation of the DNA/RNA binding protein translin (trsn) disrupts starvation-induced sleep suppression without affecting sleep or metabolic regulation under fed conditions. Targeted knockdown in approximately 30 leucokinin (Lk) neurons phenocopies trsn mutants, raising the possibility that these neurons are required for the integration of sleep and metabolic state [23].
Here, we identify a single pair of Lk neurons in the lateral horn of the fly brain that are required for the integration of sleep and metabolic state. These neurons project near the insulin-producing cells (IPCs), which are critical modulators of sleep and metabolic regulation [24][25][26]. Lateral Horn leucokinin (LHLK) neurons are dispensable for sleep under fed conditions but are required for starvation-induced sleep suppression. Functional imaging reveals that LHLK neurons have reduced activity in response to glucose application and increased activity under starved conditions. The identification of single neurons that integrate sleep and metabolic state provide a model for investigating the cellular mechanisms regulating the integration of sleep and metabolic state.

Results
Leucokinin (Lk) neuropeptide has been implicated in regulation of feeding, sleep, and circadian activity [27,28]. To determine whether Lk is required for metabolic regulation of sleep, we measured sleep under fed and starved conditions in mated female flies with disrupted Lk expression. In agreement with previous findings, driving Lk-RNAi (RNA interference) under control of Lk-galactose-responsive transcription factor (GAL4) (yeast transcription factor, Lk-GAL4>upstream activation sequence [UAS]-dicer-2 [dcr2],Lk-RNAi) significantly reduced Lk expression ( Fig 1A and 1B and S1B Fig). Control flies harboring Lk-RNAi or Lk-GAL4 transgenes alone significantly suppressed sleep during starvation, while no significant differences were detected between the fed and starved states in Lk>Lk-RNAi knockdown flies (Fig 1C and  1D). To confirm that these phenotypes are not due to off-target effects, we measured starvation-induced sleep suppression in two Lk mutants. Lk c275 is a hypomorphic allele containing a piggyBac element upstream of the Lk gene transcription start site (S1A Fig), with approximately 30% reduction in Lk levels ( Fig 1E and 1F and S1B Fig) [28]. Flies homozygous for Lk c275 failed to suppress sleep when starved (Fig 1G and 1H). We also used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) gene-editing to generate a recombinant transgenic line (Lk −/−(GAL4) ) by replacing bases 1 to 7 downstream of the Lk ATG start codon with a GAL4-element containing cassette (S1A Fig). Lk protein was not detected in the brains of Lk −/−(GAL4) mutants, confirming that this genetic modification resulted in a robust reduction of Lk function (Fig 1I and 1J and S1B Fig). Sleep while fed did not differ between Lk −/−(GAL4) lines and w 1118 controls, suggesting that Lk is not required for sleep regulation under fed conditions (Fig 1K and 1L). Conversely, control w 1118 flies, but not Lk −/−(GAL4) flies, robustly suppressed sleep under starved conditions during the day and night, indicating that Lk is required for starvation-induced sleep suppression (Fig 1K and 1L). In agreement with previous findings, starvation induces hyperactivity in control flies (S1C and S1D Fig) [20,29]. Starvation did not alter waking activity in Lk c275 and Lk −/−(GAL4) flies, indicating that Lk is required for both starvation-induced changes in sleep regulation and hyperactivity (S1C and S1D Fig).
To determine whether the sleep phenotype was caused by loss of Lk, we restored Lk in the background of each mutant and measured sleep. Pan-neuronal rescue (Lk c275 ; embryonic lethal abnormal vision [elav]-GAL4/UAS-Lk) restored starvation-induced sleep suppression (S1E Fig) and starvation-induced hyperactivity (S1F Fig). Rescue flies did not differ from heterozygous controls (Lk c275 ; elav-GAL4/+ or UAS-Lk;Lk c275 /+). The GAL4 insertion in Lk −/−(GAL4) drives expression to a pattern similar to Lk antibody, providing the opportunity to restore Lk to its endogenous expression pattern (S1G  Leucokinin antibody labels the LHLK neurons and the subesophoageal ganglion Lk (SELK) neurons, as well as a number of abdominal Lk (ABLK) neurons in the ventral nerve cord (Fig  2A). To localize the population of neurons that regulate starvation-induced sleep suppression, we restricted GAL4 expression primarily to the brain through the expression of GAL80, a GAL4 repressor, in the ventral nerve cord using teashirt-GAL80 (tsh-GAL80) [30]. Expression of CD8::GFP (Lk-GAL4>CD8:green fluorescent protein [GFP];tsh-GAL80) revealed tsh-GAL80 blocks expression in all but two ventral nerve cord neurons without affecting expression in the brain (Fig 2B). Silencing the remaining Lk neurons with light-chain tetanus toxin (TNT) (tsh-GAL80;Lk-GAL4>UAS-TNT) abolished starvation-induced sleep suppression, phenocopying the effects of silencing all Lk neurons (Lk-GAL4>UAS-TNT) (Fig 2C and 2D) [31]. Further, no differences in sleep were detected between groups in fed flies, and there was no effect of expressing an inactive variant of TNT light chain (impTNT) in Lk neurons (Fig 2C  and 2D). These findings suggest Lk neurons within the brain are required for sleep-metabolism interaction.
It has previously been reported that Apterous-GAL4 (Apt-GAL4) drives expression in the LHLK neurons, as well as neurons in the optic lobe and antennal mechanosensory and motor centers (AMMCs), and a small population of mushroom-body neurons (S2A Fig) [27,32]. Immunostaining with Lk antibody in Apt-GAL4>UAS-mCD8::GFP flies confirmed colocalization exclusively within the LHLK neurons (S2A and S2B Fig). To functionally assess the role of LHLK neurons, we genetically silenced LHLK neurons, as well as other non-Lk cells labeled by Apt-GAL4. Silencing neurons labeled by Apt-GAL4 (Apt-GAL4>UAS-TNT) inhibited starvation-induced sleep suppression and promoted sleep while fed, while no effects were observed in flies expressing impTNT (Apt-GAL4>UAS-impTNT) (Fig 2E). While these findings suggest a role for Apt-GAL4-labeled neurons in sleep regulation, it is possible that the phenotype is independent of Lk function.
To complement genetic silencing experiments, we sought to precisely ablate the LHLK neurons and measure their role in starvation-induced sleep suppression (Fig 3A). Multiphoton microscopy has been used in diverse genetic models for targeted ablation of neuronal cell types [33][34][35]. All adult Lk neurons are present in third-instar larvae and labeled by the Lk-GAL4. The SELK and anterior Lk (ALK) neurons are easily visualized, providing the opportunity to independently ablate these subtypes and measure the effect on adult behavior in an intact animal (Fig 3B). We selectively induced bilateral ablations of LHLK neurons or unilateral ablation of two control ALK neurons in immobilized third-instar larvae with a titanium sapphire multiphoton laser. Ablation of individual neurons could be visualized in larvae as a disruption of the GFP-labeled neuronal cell body ( Fig 3C). Following ablation, larvae were transferred back into food vials, and 5-to 7-day-old adult flies were tested for sleep under fed and starved conditions. After behavioral testing, brains were dissected and imaged to verify selective bilateral ablation of the LHLK neurons or unilateral ablation of two ALK neurons ( Fig 3F). Flies with ablated ALK neurons suppressed sleep during starvation similarly to controls ( Fig 3D and 3E). Conversely, bilateral ablation of the LHLK neurons abolished starvation-induced sleep suppression without affecting sleep while fed, revealing an essential role for the LHLK neurons in the integration of sleep and metabolic state (Fig 3D, 3E and 3F).
The finding that LHLK neurons are required for starvation-induced sleep suppression raises the possibility that the activity of Lk neurons is modulated by nutritional state. We selectively expressed a GFP-calmodulin and MP13 peptide sequence (GCaMP6m)-mCherry (UAS-Gerry) fusion protein that allows for ratiometric detection of Ca 2+ activity [36,37] in Lk neurons and measured the response to nutrients. The brains of fed or 24-hr-starved flies were imaged for GCaMP and mCherry signal ex vivo ( Fig 4A). Flies expressing Gerry in Lk neurons suppressed sleep similarly to control flies harboring Lk-GAL4 alone, indicating that expression of the Ca 2+ sensor does not affect starvation-induced regulation of sleep (S3A Fig). The GCaMP/mCherry ratio was elevated in the LHLK neurons of starved flies compared to fed controls, suggesting these neurons are more active during starvation ( Fig 4B). Conversely, no difference in the GCaMP/mCherry ratio between the fed and starved states was detected in the SELK neurons ( Fig 4C).
In mammals, the activity of some sleep-and wake-promoting neurons are directly modulated by glucose and other circulating nutrients [38,39]. It is possible that the activity of Lk neurons is modulated in accordance with feeding state by sensory detection of tastants or indirectly result upon detection of changes in circulating nutrients. To differentiate between these possibilities, the brains of fed flies were removed and treated with either glucose or the competitive inhibitor of glycolysis, 2 deoxy-glucose (2DG) [40][41][42]. Application of glucose reduced Ca 2+ activity in LHLK neurons compared to controls treated with Drosophila artificial hemolymph alone, suggesting these neurons are sensitive to circulating glucose ( Fig 4D). To verify that application of glucose was specific to LHLK neurons, we applied glucose or artificial hemolymph while measuring Ca 2+ response in SELK neurons. No significant differences in Ca 2+ activity were found between controls and glucose application (S3B Fig). Further, the combined application of 2DG and glucose increased Ca 2+ activity to levels greater than hemolymph alone, mimicking the starved state ( Fig 4D). Taken together, these findings indicate (E) Sleep profile representative of (D). Flies are placed in food tubes during day 1 (fed, gray), then transferred to agar during day 2 (starved, blue). White/black bars represent lights on and off, respectively. (F) Representative images of GFP-expressing Lk neurons post-LHLK (right) and ALK ablation (middle) and treated but nonablated controls (left). Red arrows indicate ablated neurons. White and yellow arrows indicate intact LHLK neurons and ALK neurons, respectively. Scale bar = 100 μm. All columns are mean ± SEM; � p < 0.05; �� p < 0.01; ��� p <0.001. Underlying data can be found in S1 Data. ALK, anterior leucokinin; CNS, central nervous system; DAMS, Drosophila Activity Monitor System; GAL4, galactose-responsive transcription factor; GFP, green fluorescent protein; IHC, immunohistochemistry; IR, infrared light; LHLK, Lateral Horn leucokinin; Lk, leucokinin; mCD8, membrane-tethered; nc82, neuropil marker; UAS, upstream activation sequence; ZT, Zeitgeber time. A portion of the head cuticle of a fed or 24-hr-starved adult female fly was removed, and GCaMP6m/mCherry fluorescence was recorded for 120 seconds. Fluorescence intensity scale represents the ratio range of GCaMP/mCherry ranging from 2 (max) to 0 (min). (F) GCaMP/mCherry ratio is increased in LHLK neurons during starvation (n = 30) compared to fed (n � 19, p = 0.0036) and 3-hr re-fed controls (n = 29, p = 0.047). One-way ANOVA (F [2, 75] = 6.2). Scale bar = 10 μm. Error bars for GCamMP6m/mCherry ratio during the fed versus starved state indicate SEM; � p < 0.05; �� p < 0.01; ��� p < 0.001. Underlying data can be found in S1 Data. ANOVA, analysis of variance; GCaMP6m, GFP-calmodulin and M13 peptide sequence; GFP, green fluorescent protein; gluc, glucose; LHLK, Lateral Horn leucokinin; max, maximum; min, minimum; SELK, subesophageal ganglion leucokinin; UAS, upstream activation sequence; UAS-Gerry, GCaMP6m-mCherry; 2DG, 2 deoxy-glucose. that the activity of Lk neurons are modulated in accordance with nutrient availability and support the notion that the LHLK neurons are more active during starvation, thereby suppressing sleep.
To examine whether the activity in Lk neurons is modulated by feeding state in an intact animal, we performed in vivo recordings in tethered flies ( Fig 4E). Briefly, a portion of the head capsule was removed so that the LHLK neurons were accessible. The activity of LHLK neurons was then recorded in flies that had been previously fed or starved for 24 hr. In agreement with ex vivo findings, the GCaMP/mCherry ratio was elevated in the LHLK neurons of starved flies, fortifying the notion that Lk neurons are more active during starvation (Fig 4F). Refeeding flies with standard food reduced the GCaMP/mCherry ratio 3 hr after refeeding ( Fig 4F). Taken together, these findings suggest the activity of LHLK neurons are modulated by circulating nutrient levels.
The identification of nutrient-dependent changes in activity of LHLK neurons raises the possibility that cell-autonomous nutrient sensors or signaling pathways function within Lk neurons to modulate sleep. To identify regulators of sleep that modulate the activity of Lk neurons, we expressed RNAi targeted to 28 RNAi lines encoding putative nutrient sensors or signaling pathways using Lk-GAL4 and measured starvation- Genetically restricting AMPK knockdown in flies harboring tsh-GAL80 (tsh-GAL80;Lk-GAL4>AMPKα-RNAi) also impaired starvation-induced sleep suppression (Fig 5C and 5D), suggesting AMPKα functions in the Lk neurons within the brain to regulate sleep.
To determine whether inhibition of AMPK signaling changes the activity of Lk neurons to resemble a starved-like state, we genetically expressed AMPKα-RNAi under control of Lk-GAL4 and measured neuronal activity in vivo using UAS-Gerry (Fig 5E and 5F; Lk-GAL4>UAS-AMPKα-RNAi; UAS-Gerry). Genetic disruption of AMPKα increased Ca 2+ activity in LHLK neurons of fed flies compared to flies expressing UAS-Gerry alone ( Fig 5E). The increase Ca 2+ activity phenocopies changes found in starved control flies, suggesting that the loss of AMPK increases the activity of Lk neurons, thereby suppressing sleep ( Fig 5F). Together, these findings suggest AMPK is active within LHLK neurons during the fed state, and reduced AMPK signaling during starvation increases LHLK activity.
Lk signals through a single Lk receptor (Lkr) that is highly expressed in the IPCs and the dorsal fan-shaped body (dFSB), both of which have been implicated in sleep regulation [24,[44][45][46]. To determine the role of Lkr in sleep regulation, we used CRISPR/Cas9 gene-editing to generate a recombinant transgenic line (Lkr −/−(GAL4) ) with a GAL4 element inserted 106 to 111 base pairs preceding the ATG translational start site, disrupting its function. Consistent with previous reports of Lkr expression, transgene expression in Lkr −/−(GAL4) >UAS-mCD8:: GFP flies labeled the IPCs, dFSB, and a number of other brain regions (Fig 6A). To determine the role of Lkr in sleep, we tested flies for sleep under fed and starved conditions. Lkr −/−(GAL4) flies failed to suppress sleep when starved, phenocopying loss of Lk function (Fig 6B). Restoring Lkr to Lkr mutant flies (Lkr −/−(GAL4) >UAS-Lkr) rescued starvation-induced sleep suppression (Fig 6B). The promoter-fusion R65C07-GAL4 predominantly labels the dFSB, while R67D01-GAL4 primarily labels the IPCs ( [47], Fig 6C and 6F). Silencing R65C07-GAL4labeled neurons with TNT reduced sleep in fed flies, consistent with a sleep-promoting role for  Fig 6D). However, there was no effect of Lkr knockdown in these cells (R65C07>UAS-dcr2, Lkr-RNAi), suggesting that Lk does not signal through the dFSB to modulate sleep. Conversely, genetic silencing or expression of Lkr-RNAi in R67D01-GAL4 neurons that label the IPCs abolished starvation-induced sleep suppression (Fig 6G and 6H). Together, these findings suggest Lkr function in the IPCs is required for starvation-induced sleep suppression. To validate a role for the IPCs, we selectively targeted Lkr in neurons labeled by Drosophila insulin-like peptide 2 (Dilp2) (Fig 6I). Selectively knocking down Lkr in Dilp2 neurons (Dilp2-GAL4>UAS-dcr2, Lkr-RNAi) prevented starvation-induced sleep loss, indicating that Lkr is required in Dilp2 neurons for starvation-induced sleep suppression (Fig 6J and 6K).

Discussion
Our findings reveal that Lk signals through a single Lkr to integrate sleep and metabolic state. Previous studies have identified a number of genes required for starvation-induced changes in sleep or locomotor activity, yet many of these genes have pleiotropic functions on behavior or metabolic function [20,29,48,49]. For example, the glucagon-like adipokinetic hormone (AKH) is responsible for energy mobilization, and genetic disruption of AKH induces obesity and abolishes starvation-induced hyperactivity [29,50,51]. Similarly, the circadian transcription factors clock and cycle are required for starvation-dependent regulation of behavior, and loss of function affects sleep both in fed and starved conditions [20]. Conversely, neuropeptide F functions within a subpopulation of circadian neurons and is selectively required for metabolic regulation of sleep [22]. Our findings that genetic manipulations that inhibit Lk signaling selectively disrupt starvation-induced modulation of sleep suggest that different neural mechanisms regulate sleep under basal conditions and in response to environmental perturbation.
The failure of Lk mutants to suppress sleep under starved conditions phenocopies mutation of the RNA/DNA binding protein trsn. Loss of trsn does not impact feeding behavior but impairs starvation-induced sleep suppression, suggesting that trsn is not generally required for hunger-induced behavior [23]. While trsn is broadly expressed in the fly nervous system [52,53], we previously found that selective knockdown of trsn in Lk neurons disrupted starvation-induced sleep suppression [54]. These findings raise the possibility that trsn functions to regulate changes in the physiology of Lk neurons to modulate sleep under starved conditions. A central question is how Lk neurons modulate numerous complex behaviors and physiological processes. In adults, Lk is expressed in four pairs of neurons in the brain and 11 pairs in the ventral nerve cord, which regulate diverse behaviors and physiological processes [55][56][57]. Recent work suggests the ABLK neurons in the thoracic ganglion are critical regulators of water consumption and contribute to the altered stress resistance and water content in Lk mutant flies [46]. The SELK neurons connect the gustatory receptors to the subesophageal ganglia and ventral nerve cord. Although a specific function has not been identified to SELK neurons, silencing of all Lk neurons disrupts gustatory behavior, and a mutation in the Lk locus affects meal size [28,58], raising the possibility that these behaviors are regulated by SELK neurons. Lastly, the LHLK neurons project to the superior lateral protocerebrum, medial protocerebrum, and peduncle and axonal stalk of the mushroom bodies [56]. The LHLK UAS-Gerry flies (blue and white, n = 8) compared to flies harboring Gerry alone (blue, n = 8, p = 0.44, t = 0.78), Unpaired t test. Fluorescence intensity scale represents the ratio range of GCaMP6m/mCherry ranging from 2 (max) to 0 (minimum). Scale bar = 10 μm. Error bars for GCamp6m/mCherry ratio during the fed versus starved state indicate SEM; � p < 0.05; �� p < 0.01; ��� p < 0.001. Underlying data can be found in S1 Data. AMPK, 5 0 adenosine monophosphate-activated protein kinase; ANOVA, analysis of variance; GAL4, galactose-responsive transcription factor; GCaMP6m, GFP-calmodulin and M13 peptide sequence; GFP, green fluorescent protein; LHLK, Lateral Horn leucokinin; Lk, leucokinin; max, maximum; RNAi, RNA interference; tsh, teashirt; UAS, upstream activation sequence; UAS-Gerry, GCaMP6m-mCherry; ZT, Zeitgeber time. Single-neuron regulation of sleep-metabolism interactions neurons we identify here as regulating sleep-metabolism interactions receive inputs from clock neurons and are thought to modulate locomotor activity and sleep [27]. Supporting the notion that LHLK neurons are outputs of the clock, previous work has found that silencing Lk within these neurons attenuates circadian rhythms [27]. Conversely, we find that silencing of these neurons has little effect on sleep yet abolishes starvation-induced sleep suppression. Together, these findings support a role for LHLK neurons in sleep regulation, yet discrepancies remain about the conditions under which these neurons regulate sleep.
Lk neurons have also been implicated in the modulation of sleep following the ingestion of a meal. Postprandial sleep is enhanced in Lk-deficient flies and reduced in flies with thermogenetically activated Lk neurons sleep, whereas Lk knockdown increases the probability of falling asleep after a meal [59]. Similarly, we found that knockdown of Lk in Lk-expressing neurons results in increased sleep during starvation. In contrast, it was previously reported that Lk signals the fan-shaped body neurons to regulate postprandial sleep [59]. These results raise the possibility that distinct circuitry regulating starvation-induced sleep suppression and postprandial sleep, which could be influenced by distinct time-scales at which both behaviors are being executed. In addition, it is possible that a distinct subpopulation of Lk neurons is responsive to individual nutrients in comparison to starvation itself, which is sensed by LHLK neurons. Taken together, these findings suggest different neural mechanisms underlie Lkdependent regulation of postprandial feeding and circadian rhythms.
The Drosophila genome encodes for a single Lk target, the Lkr, that is expressed in the lateral horn, the ventral nerve cord, the IPCs, and the sleep-promoting fan-shaped body [27,28,59]. The fan-shaped body, a subregion of the Drosophila central complex, is a primary sleep-promoting region [45,60], while the IPCs are proposed integrators of sleep and feeding state [61]. Previous studies suggest that Lkr function within the fan-shaped body is required for proper regulation of circadian rhythms and postprandial sleep [27,59]. Here, we find that targeted knockdown of Lkr within the IPCs phenocopies LHLK ablation, suggesting the LHLK neurons signal to the IPCs to modulate starvation-induced regulation of sleep. Supporting these findings, recent work has shown that Lk and Lkr mutants display increased levels of Dilp2 and Dilp3 immunoreactivity in the IPCs [46]. However, anterograde trans-synaptic labeling revealed no direct synaptic inputs between Lk neurons and the IPCs, raising the possibility that that Lk inputs to the IPCs could occur via paracrine signaling [46]. Taken together with previous studies, these findings suggest different Lk targets regulate postprandial feeding, circadian rhythms, and sleep or that Lk functions through paracrine signaling to modulate different targets.
In Drosophila, a number of circulating nutrients including fructose, trehalose, and glucose have been found to affect central brain physiology and behavior [42,62,63]. While nutrients may be detected by gustatory receptors expressed in the periphery to regulate sleep [9,64], sugar receptors and transporters are also expressed in the brain [65]. The identification of suppression is absent with Lkr knockdown in Lkr-expressing neurons (R67D01, n = 41, p = 0.53). Control UAS-Lkr-RNAi/+ (n = 25, p < 0.0001) and dcr2,R67D01-GAL4 (n � 53, p < 0.0001) suppress sleep in response to starvation. Two-way ANOVA (F [2, 232] = 13.86). (J) Knocking down Lkr in the IPCs (Dilp2-GAL4>UAS-dcr2,Lkr-RNAi, n � 30, p = 0.67) results in flies that fail to suppress sleep in response to starvation, while dcr2,Dilp2-GAL4/+ control flies suppress sleep (n = 25, p = 0.0016). Two-way ANOVA (F [1, 108] = 4.1). (K) Sleep profile representative of (J). Flies are placed in food tubes during day 1 (fed, gray), then transferred to agar during day 2 (starved, blue). White/black bars represent lights on and off, respectively. All columns are mean ± SEM; � p < 0.05; �� p < 0.01; ��� p < 0.001. Underlying data can be found in S1 Data. ANOVA, analysis of variance; CD8::GFP, membrane-tethered GFP; LK-GAL4>CD8:GFP;tshGAL80; dcr2, dicer-2; dFSB, dorsal fan-shaped body; Dilp2, Drosophila insulin-like peptide 2; GAL4, galactose-responsive transcription factor; GFP, green fluorescent protein; impTNT, inactive variant of tetanus toxin; IPC, insulinproducing cell; Lk, leucokinin; Lkr, leucokinin receptor; nc82, neuropil marker; RNAi, RNA interference; TNT, tetanus toxin; tsh, teashirt; UAS, upstream activation sequence; ZT, Zeitgeber time. LHLK neurons as being active under starvation conditions and suppressed by glucose provide a system to investigate feeding-state-dependent changes in neural activity. A number of neurons in the fly brain are acutely regulated by feeding state, including the starvation-active Taotie neurons that inhibit IPCs of the pars intercerebralis to regulate insulin-like peptide release under nutrient deprivation conditions [66][67][68]. Conversely, the IPCs function as cell-autonomous nutrient sensors that are activated by glucose through the inhibition of K ATP channels [69]. Further, the LHLK nutrient phenotype is similar to the neurons within the ellipsoid body labeled by the sodium/glucose co-transporter SLC5A11 that are active during starvation and promote feeding [41,70]. SLC5A11 and its cognate neurons are required for a variety of hunger-induced feeding behaviors, but the effect on sleep has not been identified [70]. Our screen found that knockdown of SLC5A11 in Lk neurons did not affect starvation-induced sleep suppression, suggesting alternative regulators of sleep. The identification of LHLK neurons as starvation-active neurons provides a system for identification of additional nutrient sensors that regulate sleep.
AMPK functions as a cell-autonomous regulator of energy allocation and induces physiological changes associated with starvation [71,72]. AMPK consists of a heterotrimeric complex that is activated by AMP and modulates diverse intercellular signaling pathways, including mammalian target of rapamycin (mTOR), forkhead box (FoxO), and sirtuin-1 (SIRT1) [73]. Canonically, AMPK is activated during starvation and increases neuronal activity, though this effect varies by neuronal subtype [74,75]. For example, in Caenorhabditis elegans, starvationinduced AMPK activation leads to inhibition of neurons that modulate local search behavior in response to food deprivation while promoting activity in neurons that trigger dispersal behavior [76]. Here, we find that knockdown in LHLK neurons using multiple independently derived RNAi lines results in flies that reduce sleep under the fed state and increases the activity of LHLK neurons, similar to neural activity seen during the starved state. Ubiquitous disruption of AMPK in Drosophila induces hypersensitivity of the locomotor response to starvation and reduces starvation resistance [74]. Conversely, we find that selectively disrupting AMPK function in Lk neurons promotes starvation-induced hyperactivity and sleep loss during the fed state, suggesting a neural-circuit-specific function for AMPK. While our findings suggest that AMPK functions as an important modulator of LHLK neuronal activity and statedependent changes of activity within LHLK neurons, it is also possible that AMPK generally modulates the activity of Lk neurons, resulting in sleep loss. The findings that the activity of SELK neurons is not elevated during starvation raises the possibility of neuron-specific AMPK function.
Taken together, the identification of LHLK neurons as critical modulators of sleep-metabolism interactions provides a system for identifying novel nutrient-sensing and signaling mechanisms that modulate sleep. These findings illustrate the need to determine how Lk neurons modulate different aspects of sleep regulation, including their reported role as circadian output neurons, in regulation of sleep-metabolism interactions, and in postprandial sleep regulation [23,27,59]. Further investigation of feeding-state-dependent changes in Lk signaling and the identification of neuronal inputs and targets of LHLK neurons will provide mechanistic insight into how animals integrate sleep with changes in their internal and external environments.

Generation of GAL4 knock-in mutants and UAS-Lk
Lk −/−(GAL4) and Lkr −/−(GAL4) were generated by Wellgenetics (Taipei City, Taiwan) using the CRISPR/Cas9 system to induced homology-dependent repair (HDR) using one guide RNA  . At the gRNA target site, a donor plasmid was inserted containing a GAL4:: VP16 and floxed 3xP3-RFP cassette [82]. For Lk −/−(GAL4) , following the translational ATG start site, bases 1 to 7 were replaced by the knock-in cassette. For Lkr −/−(GAL4) , preceding the ATG start site, bases 111 to 106 were replaced by the knock-in cassette. All lines were generated in the w 1118 background [77]. Proper insertion loci for both mutations were validated by genomic PCR.

UAS-Lk
The

Behavioral analysis
The DAMS detects activity by monitoring infrared beam crossings for each animal [84]. These data were used to calculate sleep information by extracting immobility bouts of 5 minutes using the Drosophila Counting Macro [85,86]. For experiments examining the effects of starvation on sleep, flies were kept on a 12:12 LD cycle. Mated female flies were briefly anesthetized with CO 2 and placed into plastic tubes containing standard food. All flies were given 24 hr to recover after being anesthetized. Activity was recorded for 24 hr in food tubes prior to transferring flies into tubes containing 1% agar diluted in dH 2 O (Fisher Scientific) at Zeitgeber time (ZT) 0. Activity was monitored for an additional 24 hr on agar. For the screen, percent change in sleep during starvation was calculated as the sleep duration on agar minus the sleep duration in food tubes, divided by the sleep duration in food tubes for each fly assayed multiplied by a hundred [11,54].

Immunohistochemistry
The brains of 5-to 7-day-old female flies were dissected between ZT 4-ZT 9 in ice-cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde, PBS, 0.5% Triton-X for 30 minutes as previously described [87]. Brains were then rinsed 3× with PBS, 0.5% Triton-X (PBST) for 10 minutes and overnight. In the following day, brains were incubated for 24 hr in primary antibody (

Blue-dye assay
Briefly, flies were maintained on standard fly food. At ZT 0, flies were transferred to vials containing 1% agar, 5% sucrose, and 2.5% blue dye (FD&C Blue Dye No. 1). Following 30 minutes of feeding, flies were flash frozen on dry ice, and four flies were homogenized in 400 μL PBS (pH 7.4, Thermo Fisher Scientific) per sample. Color spectrophotometry was then used to measure absorbance at 655 nm in a 96-well plate reader (iMark; Millipore Sigma, Burlington, MA, USA). Baseline absorbance was determined by subtracting the absorbance measured in non-dye-fed flies from each experimental sample.

Functional imaging of Lk neurons
Five-to seven-day-old female flies were collected and placed in vials containing fresh food (fed) or a wet KimWipe paper (starved) for 24 hr. All experiments were done between ZT 4-ZT 7 to account for rhythmic excitability of Lk neurons [27]. For imaging brain explants, previously established methods for calcium imaging were used with modifications [42,65]. Brains of fed or 24-hr-starved flies were dissected and placed in glass wells (Pyrex, Corning, Corning, NY, USA) containing artificial hemolymph (140 mM NaCL, 2 mM KCl, 4.5 mM MgCl2, 1.5 mM CaC2, and 5 mM HEPES-NaOH with pH 7) and allowed a 5-minute recovery period before being recorded. For 2DG experiments, fed brains were dissected and placed in 400 mM 2DG (Sigma Aldrich) in artificial hemolymph, 200 mM glucose (Sigma Aldrich) in artificial hemolymph, or artificial hemolymph alone for a total of 70 minutes. Every 20 minutes, solutions were exchanged. Coverslips were treated with poly-L-lysine (Sigma Aldrich) to ensure that brains were in the same position during imaging and placed onto chamber (RC-21BBDW; Warner Instruments, Hamden, CT, USA). Fly brains were bathed in artificial hemolymph solution and imaged using a 20× air objective lens on an inverted confocal microscope (Nikon A1R on a Ti-E inverted microscope). The pinhole was opened to 244.43 μm to allow a thicker optical section to be monitored. UAS-GCaMP-R (GCaMP and mCherry) was expressed in Lk neurons and simultaneously excited with wavelengths of 488 nm (FITC) and 561 nm (TRITC). Recording were taken for 120 seconds, capturing 1 frame/5 seconds with 512 × 512 resolution. For analysis, regions of interest (ROIs) were drawn manually, capturing the same area between experimental and control. The mean fluorescence intensity was subtracted from background mean fluorescence intensity for FITC and TRITC per frame. Then, the ratio of GCaMP6.0 to mCherry was calculated and plotted as an average of the total time recorded per brain imaged. In vivo imaging was performed using a previously described protocol with some modifications [89,90]. Briefly, fed, 24-hr-starved, or 3-hr re-fed (standard Bloomington Recipe) flies were anesthetized on ice and secured in a 200-μL pipette tip with head and proboscis accessible. The pipette tip was placed in a small chamber at an angle of 140˚, allowing the dorsal and posterior surface of the brain to be imaged. A small hole was cut in the tin foil and fixed to the stage and fly head, leaving a window of cuticle exposed, then sealed using dental glue (Tetric EvoFlow; Ivoclar Vivadent, Schaan, Lichtenstein). The proboscis was extended, and dental glue was used to secure it in place, ensuring the same position throughout the experiment.
A 21-gauge 1 1/4 needle (PrecisionGlide; Becton Dickinson, Franklin Lakes, NJ, USA) was used to cut a window in the fly cuticle. A drop of artificial hemolymph was placed on the cuticle, and the connective tissue surrounding the brain was dissected. Flies were allowed to recover from the procedure for 30-45 minutes in a humidified box. Mounted flies were placed under a confocal microscope (Nikon A1R on an upright microscope) and imaged using a 20× water-dipping objective lens. The pinhole was opened to 244 μm to allow a thicker optical section to be monitored. The settings and data analysis were performed as described above.

Targeted multiphoton ablation of Lk neurons
Female third-instar larvae expressing GFP in Lk neurons were selected and anesthetized in ethyl ether (Thermo Fisher Scientific, E134-1) for 2-5 minutes. Larvae were placed dorsally on a microscope slide, and a coverslip was placed on the larvae. Ringer's solution was applied onto the larvae below the coverslip. Larvae were imaged using a 25× water-dipping objective lens on a multiphoton microscope (Nikon A1R) containing a Chameleon Vision II Ti:Sapphire tunable laser. Excitation laser light of 870 nm was used. Images were acquired at 1 frame per second with a resolution of 512 × 512 pixels. For each neural ablation, a total of four frames were acquired. Two frames were captured prior to ablation for a duration of approximately 2 seconds, followed by ROI stimulation of 2-4 seconds and two frames after ablation. Following ablations, larvae were placed in vials containing food and allowed to grow. Sleep in food tubes and on agar was measured 5-7 days posteclosion in the DAMS. In order to verify which neurons were ablated after behavioral assay, flies were anesthetized on ice, and the central nervous system (CNS) was dissected. Fly CNS was fixed in 4% paraformaldehyde, 0.5% PBST for 30 minutes. Following fixation, samples were imaged in 2-μm sections with a Nikon A1R confocal microscope (Nikon) using a 20× oil immersion objective. Ablations that resulted in the formation of supernumerary neurons or deletions of two different subpopulations of Lk neurons were removed from analysis.